Cancer is a leading cause of cancer-related deaths in the United States. For example, colorectal cancer resulted in 51,370 deaths during 2010. Despite this prevalence, therapeutic options for cancer are currently limited to surgery, radiation, or chemotherapy. These non-specific therapies are effective for early stage but not for metastatic cancer and frequently cause untoward gastrointestinal and hematologic side effects. Further, monoclonal antibody treatments specific for cancer are reserved for patients with advanced disease because these therapies provide limited clinical benefit, are cost prohibitive and require intravenous delivery. Thus, there is a major unmet medical need for the development of selective, inexpensive and non-toxictargeted treatments for human cancer.
Therapies targeting protein components of dysregulated signal transduction pathways can be efficacious anti-cancer therapies with minimal adverse effects. The initiation and progression of colon, breast, prostate, pancreatic and other human tumors has been shown to depend on mutations in β-catenin (β-cat) itself or upstream regulators, leading to stabilization and increased levels of β-cat that in turn activate genes involved in tumor maintenance and proliferation. Direct inhibition of Wnt/β-cat is an oncology target but simply blocking the ubiquitously expressed β-cat, however, has adverse consequences on cell adhesion and tissue integrity in normal tissues. Compounds that potently and selectively inhibit the nuclear availability of TCF proteins that binds β-cat and is required for its activation of downstream Wnt target genes in a number of human cancers could be a useful therapeutic. Compounds that selectively inhibit the Wnt pathway very far downstream of normal physiological function of the Wnt pathway could decrease human cancer cell proliferation in vitro and inhibit cancer proliferation in vivo. Further, compounds that work very far downstream of normal physiological function of Wnt will work with minimal side effects, thus showing novel therapeutic utility. These targeted Wnt inhibitors may also be used in combination with traditional cancer chemotherapies including 5-fluoruracil, oxaliplatin, or irinotecan. Combination therapy of Wnt molecular pathway inhibitors with chemotherapeutics will allow for lower effective doseages resulting in fewer adverse side effects.
Certain efforts have focused with limited success on decreasing cellular pools of β-cat because of decreased adhesion and tissue integrity in normal tissues. A compound that inhibits the Wnt pathway far downstream and remote of β-cat could potently and selectively increasephosphorylated TCF proteins in the nucleus without affecting β-cat levels outside the nucleus. Accordingly, the effects on normal tissue would be minimal. A compound that potently and selectively inhibits β-cat availability in the nucleus could decrease cellular proliferation in cancer. This is in contrast to the well-established Wnt inhibitors of the multi-protein destruction complex (e.g., IWR-1), that have no effect on colorectal and other cancer cell proliferation. Thus, a compound that selectively inhibits Wnt by increasing TCF protein phosphorylation would possess several characteristics that render them distinct and superior to other reported Wnt inhibitors: i). inhibition of the Wnt pathway at the transcriptional level provides not only inhibition of Wnt target genes but also preserves upstream Wnt signaling, ii). they would not interfere with cellular adhesion and result in maintenance of normal tissue structure in contrast to most other previously reported Wnt inhibitors. Targeting TCF provides several advantages over other target proteins in the Wnt pathway because its action is at the nuclear level similar to transcriptional co-activator antagonists. Targeting TCF also blocks the Wnt pathway completely. Phosphorylation of TCF proteins inhibits transcriptional activities of them. TCF4 and LEF1 are transcriptional activators and stimulation of phosphorylation results in inhibition of transcription. HIPK2 is the kinase that does this phosphorylation and thus, developing activators of HIPK2 provides a rapid and efficient means of identifying potent anti-cancer agents and compounds that could decrease the proliferation of cancer and cancer stem cells.
The Wnt pathway is dysfunctional in several other cancers including colon cancer, prostate cancer, breast cancer, ovarian cancer, uterine cancer, liver cancer, malignant melanoma, pancreatic cancer and gastric cancer and glioblastoma and other brain cancers. Developmental work has been done in several institutions to capitalize on the promise of Wnt inhibitors in cancer but problems associated with interference of normal physiological function of Wnt has not been addressed. Thus, the invention disclosed herein identifies and validates new paradigms to identify new biological pathways and targets that can be used to inhibit cancer cell growth and proliferation.
In addition to cancer, the Wnt pathway plays a pivotal role in neurochemistry and is involved in neurogenesis and neuronal disease. As such, small molecules described herein may modulate neuronal diseases and be of utility in inhibiting diseases of neurological origin.
Emerging evidence suggests that Wnt signaling regulates crucial aspects of a number of cancer tumor initiation and progression. The Wnt pathway is dependent on the cellular level of β-cat. Under normal physiologic conditions, the majority of β-cat resides in the cytoplasm and is maintained at a low level through degradation that is regulated by a multi-protein “destruction” complex containing axin. Upon Wnt stimulation, Axin translocates to the cell membrane to interact with LRP5 and Dvl. Dvl becomes phosphorylated and subsequently inhibits GSK3β-kinase phosphorylation of β-cat thereby resulting in the accumulation of non-phosphorylated β-cat in the cytoplasm. This non-phosphorylated β-cat then translocates to the nucleus where it interacts with the TCF/LEF family of transcription factors, binds to Wnt DNA response elements of target genes, and recruits the transcriptional machinery to enhance gene expression. The Wnt target genes include genes that promote cellular proliferation, metabolism, cellular migration, and differentiation. Additionally, several protein components of the Wnt pathway are frequently mutated and/or over-expressed in many types of cancer. β-cat and several protein components of the multi-protein degradation complex including APC and Axin2 have been found to be mutated in patients with colon cancer. These mutations all prevent β-cat degradation leading to enhanced cellular β-cat levels and subsequent activation of Wnt target gene expression. β-cat (non-mutated) is also over-expressed in several cancers. Recent studies have also highlighted the importance of alternatively-spliced TCF isoforms in cancer. These TCF isoforms have divergent effects on Wnt target gene activity. For example, truncated TCF isoforms often cannot interact with β-cat and thus, act as dominant negative transcription factors blocking recruitment of β-cat and the associated transcriptional machinery to the Wnt gene promoters. In contrast, another TCF isoform that has a novel activation function leads to activation of Wnt target genes. An analysis of TCF isoform expression in both normal and cancerous colon tissue indicated that relative TCF isoform expression changes with tumor progression influence the Wnt target gene activity. As an attractive oncology target, several new small molecules, existing drugs, natural compounds and biologics have been reported to act as inhibitors of the Wnt signaling pathway. These agents either act as β-cat/TCF antagonists, molecules stabilizing the multi-protein destruction complex (i.e., IWR-1) transcriptional co-activators modulators, Dvl PDZ-domain antagonists, or other unknown targets. But these agents cause toxicity. Compounds targeting the Wnt pathway far downstream should avoid the untowardside effects from influencing the cytoplasmic effects of the Wnt pathway and focus on inhibiting only the transcription of Wnt target genes leading to decreased cellular proliferation and migration. Such a compound is 1 and refined analogs.
Compound 1 inhibited the canonical Wnt pathway independently of β-cat. This is apparent because: a) 1 does not affect β-cat levels or localization, b) 1 potently blocks β-cat activity induced either by GSK3β inhibitor BIO, by a mutation in APC in SW480 cells, or by over-expression of constitutive active form of β-cat, and c) 1 did not affect interaction of β-cat with TCF proteins or co-activators (CBP and p300). Together, these results indicated that 1 inhibited canonical Wnt signaling independent of β-cat stabilization, localization, and its interaction with its co-activators. This is in contrast to previously characterized Wnt inhibitors that block various points in the signaling pathway. Instead of affecting β-cat, 1 inhibited the Wnt pathway through HIPK2 activation based on the following observations; a) 1 caused a mobility shift of over-expressed TCF3, LEF1, and TCF4, that are phosphorylated by HIPK2, and 1 induced phosphorylation of TCF3, b) 1 induced a mobility shift of HIPK2 and siRNA for HIPK2 rescued inhibition of Wnt reporter gene expression by 1, indicating that 1 exerts its activity through HIPK2, c) consistent with previous observation in which HIPK2 phosphorylated TCF proteins to prevent their association with β-cat and chromatin, 1 decreased recruitment of TCF4 to its target site in the endogenous c-MYC promoter, and d) over-expression of HIPK2 enhanced 1 activity on the Wnt pathway. Consistent with their modes of action (TCF4 is an activator whereas TCF3 is a repressor)—the Wnt reporter responded by decreasing (for TCF4) and increasing (TCF3) luciferase activity by 1; combination with HIPK2 further enhanced this interaction. Finally, the physiological relevance for compound 1 induced HIPK2 activation during development was confirmed by evaluating anterior/posterior marker genes expression. Vent2 and Otx2 are regulated by TCF3 during anteroposterior axis specification in xenopus embryo and HIPK2 antagonizes them via phosphorylation of TCF3. Xenopus embryos incubated with compound 1 showed reduced anterior marker gene Otx2, whereas anterior and ventral expression domain of Vent2 was expanded. These phenotypes are very similar to embryos with TCF3-knockout and contrast to those of HIPK2-knockout. Together, the observations show that compound 1 activates HIPK2 to remove TCF proteins from their target DNA via phosphorylation.